CN110305140A - Pyrrolin miazines selectivity JAK2 inhibitor - Google Patents

Pyrrolin miazines selectivity JAK2 inhibitor Download PDF

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CN110305140A
CN110305140A CN201910698079.2A CN201910698079A CN110305140A CN 110305140 A CN110305140 A CN 110305140A CN 201910698079 A CN201910698079 A CN 201910698079A CN 110305140 A CN110305140 A CN 110305140A
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compound
pyrrolin
miazines
selectivity
pharmaceutically acceptable
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CN110305140B (en
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郑永勇
魏农农
金华
周峰
黄美花
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CHENGDU JINRUI JIYE BIO-TECHNOLOGY CO LTD
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Shanghai Xunhe Pharmaceutical Technology Co Ltd
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Priority to CN201910698079.2A priority Critical patent/CN110305140B/en
Publication of CN110305140A publication Critical patent/CN110305140A/en
Priority to EP19939211.9A priority patent/EP4006031A4/en
Priority to PCT/CN2019/127676 priority patent/WO2021017384A1/en
Priority to CA3153437A priority patent/CA3153437A1/en
Priority to GB2204901.9A priority patent/GB2603386B/en
Priority to AU2019459552A priority patent/AU2019459552A1/en
Priority to JP2021578190A priority patent/JP7335644B2/en
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Priority to US17/581,178 priority patent/US20220144844A1/en
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Abstract

The invention belongs to biomedicine technical fields, and in particular to a kind of pyrrolin miazines selectivity JAK2 inhibitor or its pharmaceutically acceptable salt.Compared with prior art, azolopyrimidines, its stereoisomer and its pharmaceutically acceptable salt provided by the invention have better Janus kinase inhibiting activity, and it inhibits target spot to be selectively significantly better than existing compound JAK2, and preferred compounds of the invention shows good pharmacokinetic property, has the potentiality for being developed into selective JAK2 inhibitor.

Description

Pyrrolin miazines selectivity JAK2 inhibitor
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of pyrrolin miazines selectivity JAK2 inhibition Agent and its pharmaceutically acceptable salt.
Background technique
JAK, that is, Janus Kinase (Janus kinases) is a kind of non-receptor type tyrosine protein kinase (PTK).JAK- STAT access is mainly made of four parts: (1) the extracellular signal factor；(2) receptor；(3) jak kinase；(4) signal transduction and turn It records activator protein (STAT).JAK-STAT is the most important signal pathway other than second messenger system.Jak kinase passes through combination Receptor experiences extracellular signal, such as interferon, interleukins, growth factor, and transmits information to STATs.Phosphorylation STATs can be transferred to nucleus from intracellular.And every kind of different STAT is integrated to different promoter DNA sequence On.Promoter can control the expression of its DNA sequence dna, and DNA transcription is caused to change with activity level, and then the growth of influence cell, The elementary cells function such as differentiation and death.
The albumen of jak kinase family shares 4, including JAK1, JAK2, JAK3, TYK2.From the acquired expression of function or dash forward Variation analysis from the point of view of, JAK1, JAK3 are more related with immunological regulation, JAK2 then with the direct phase of the generation of red blood cell and blood platelet It closes.From the point of view of afunction analysis, it is lethal that JAK1, JAK2 afunction will cause mouse embryo, not yet finds in human body Perhaps, the relevant disease of JAK1, JAK2 afunction, shows the importance of JAK1/2 physiological function indirectly.JAK3 afunction It will cause serious combined immune deficiency, this is also the targeting JAK3 being subsequently noted, to adjust autoimmune-associated diseases Foundation.The functional study of TYK2 is less, has been reported that it can cause defect relevant to innate immune.
The exploitation of JAK2 inhibitor is greatly facilitated in discovery of the JAK2V617F mutation in myeloproliferative tumour (MPN). MPN is one group of chronic disease characterized by abnormal hematopoiesis progenitor cell proliferation in marrow.MPN includes myelofibrosis (myelofibrosis, MF), polycythemia vera (Polycythemia vera, PV), primary thrombocytosis (EssentialThrombocythemia, ET) and chronic myelocytic leukemia (Chronic MyelogenousLeukemia, CML).About 95% PV patient and MF the and ET patient of 50-60% have had been found that JAK2V617F Single amino acid mutations, cause JAK2 conformational change, causes the sustained activation for not depending on the kinase region of extracellular cytokine signaling, and then cause cell Hyperplasia and hematologic cancers.
The Ruxolitinib of WO2007070514A report is initially developed by Incyte, is a JAK1/JAK2 small molecule Kinase inhibitor.FDA approval is obtained in November, 2011, for treating the myelofibrosis MF of middle and high danger.2014 further It is granted to be used for polycythemia vera.Ruxolitinib, which can reach, alleviates spleen increase caused by JAK2V617F is mutated, Mitigate the symptom of patient's weakness.
Ruxolitinib can not reduce the JAK2V617F mutational load of variation blood cell, so Ruxolitinib Curative effect can hardly be brought.Not high additionally, due to the JAK2 target spot selectivity of Ruxolitinib, side effect is obvious, The toxic side effect of Ruxolitinib mainly includes anaemia, thrombopenia, neutrophilic granulocytopenia and diarrhea etc..
There is inflammatory syndrome apparent, that prognosis is poor after being discontinued in the report display Ruxolitinib of early stage, subsequent In follow-up in 3 years, lasting similar adverse reaction is not observed, prompting such reaction may be deactivated Ruxolitinib Caused serious withdrawal inflammatory syndromes, should monitor the size of spleen closely, if the spleen during Ruxolitinib treatment It is dirty still have grow up, the related symptoms of MF are possible to revert to baseline level or even continue to progress after drug withdrawal.Therefore when consideration is interrupted When Ruxolitinib is treated, dosage should be gradually decreased or merging uses corticosteroid therapy.
The MPN drug development of a new generation focuses on the inhibitor of JAK2 selectivity, it is desired to be able to reduce due to targeting Increase curative effect caused by JAK1 while excessive side effect.
Currently, a series of patent application of JAK inhibitor has been disclosed, as CN101370792A, WO2010017122, CN101421250A, WO2010074947A1 etc..Although it is disclosed that a series of JAK inhibitor, but still need to that exploitation is new to be had The JAK inhibitor class compound of more preferable drug effect, more low side effect, especially JAK2 selective depressant.
Summary of the invention
In order to overcome the problems of in the prior art, the purpose of the present invention is to provide a kind of pyrrolin and pyrimidines Class JAK2 selective depressant.
To achieve the goals above and other related purposes, the present invention adopts the following technical scheme: it is a kind of shown in formula I Pyrrolin miazines compound, its pharmaceutically acceptable salt:
Wherein:
X is O or is not present；
Y is O, S, SO2Or NR；
W is N or CH；
R is hydrogen, C1~6Alkyl, C2~6Alkenyl, C2~6Alkynyl, C1~6Alkoxy or C1~6Carbonyl；
M is 0,1,2,3,4,5 or 6；
N is 0,1 or 2.
Preferably, in which:
X is O or is not present；
Y is O, S, SO2Or NR；
W is N or CH；
R is hydrogen or C1~4Alkyl；
M is 0,1,2 or 3；
N is 0 or 1.
More preferred, in which:
X is O or is not present；
Y is O, S, SO2Or NR；
W is N or CH；
R is hydrogen or C1~3Alkyl；
M is 0,1 or 2；
N is 0 or 1.
Further, in which:
X is O or is not present；
Y is O, S, SO2Or NR；
W is N or CH；
R is hydrogen or methyl；
M is 0,1 or 2；
N is 0 or 1.
Typical compound of the invention includes, but are not limited to following table 1 compound:
Table 1
The second object of the present invention is the provision of the synthetic method of above compound:
(1) compound 1 and 2 is made 3 through condensation reaction；
(2) compound 3 aoxidizes to obtain compound 4 through potassium hydrogen persulfate；
(3) compound 4 obtains compound 5 through sodium borohydride reduction；
(4) the obtained versatile intermediates 7 of cyclization reaction occur after sulfonyl methane chlorine activation for the hydroxyl of compound 5；
(5) compound 7 is reacted with general formula IA compound condensation is made final product I；
In step (5) described in each group text as defined above.
The third object of the present invention be to provide above compound as novel JAK inhibitor preparation prevention or treatment with Purposes in the drug of JAK related disease is specifically primarily referred to as preventing or treating following disease: the disease of immune system, packet Include organ-graft refection (such as allosome inhibits repulsion and graft versus host disease)；Autoimmune disease, including such as lupus, Multiple sclerosis, adolescent arthritis, psoriasis, ulcer disease colitis, Crohn's disease, is exempted from rheumatoid arthritis self Epidemic disease thyroid disease etc.；Skin disease, including such as ox-hide moss, skin itch, atopic dermatitis: allergic conditions, including for example Asthma, rhinitis etc.；Viral disease, including such as hepatitis B, hepatitis C, varicella virus；I type glycosuria Disease and diabetic complication；Alzheimer disease, scheroma, myelofibrosis, piastrenemia, polycythemia or white Blood disease, Huppert's disease；Cancer, including such as solid tumor (such as prostate cancer, kidney, liver cancer, film gland cancer, gastric cancer, mammary gland Cancer, lung cancer, head-neck carcinoma, thyroid cancer, glioblastoma, melanoma etc.), cutaneum carcinoma (such as skin T cell lymphoma, skin Skin son cell lymphoma) etc..
Derivative of the invention can pass through the sides such as oral, injection in implementing treatment of diseases with the formation of composition Formula, for treating associated cancer and other diseases.When for taking orally, conventional solid pharmaceutical preparation such as tablet, powder can be prepared into Agent or capsule etc.；When for injecting, injection can be prepared into.
The fourth object of the present invention is to provide a kind of composition, and the composition includes the above-mentioned pyrroles of therapeutically effective amount Miazines compound, its stereoisomer, its pharmaceutically acceptable salt and pharmaceutically acceptable carrier.
Pharmaceutically acceptable salt, for example, it can be mentioned that metal salt, connect salt, with organic base formed salt, with inorganic acid shape At salt, the salt formed with organic acid, the salt etc. that is formed with alkalinity or acidic amino acid.The non-limiting example packet of metal salt Include but be not limited to salt of alkali metal, such as sodium salt, sylvite etc.；Salt of alkaline-earth metal, such as calcium salt, magnesium salts, barium salt, aluminium salt etc.. The non-limiting example of the salt formed with inorganic acid includes but is not limited to be formed with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid etc. Salt.With organic acid formed salt non-limiting example include but is not limited to and formic acid, acetic acid, trifluoroacetic acid, fumaric acid, grass The salt of the formation such as acid, malic acid, maleic acid, tartaric acid, citric acid, succinic acid, methanesulfonic acid, benzene sulfonic acid, p-methyl benzenesulfonic acid.
The carrier addressed refers to the carrier of pharmaceutical field routine, such as: diluent, excipient such as water；Adhesive is such as fine Tie up plain derivative, gelatin, polyvinylpyrrolidone etc.；Filler such as starch etc.；Burst apart agent such as calcium carbonate, sodium bicarbonate；In addition, Other adjuvants such as flavouring agent and sweetener can also be added in the composition.
The various dosage forms of composition of the invention can be prepared using the method for medical domain routine, wherein it is active at The content divided is 0.1%~99.5% (weight ratio).
Amount of application of the invention can be according to route of administration, the age of patient, weight, the type for the disease treated and serious Degree etc. is changed, and daily dose is 0.001-30mg/kg weight (oral) or 0.005-30mg/kg weight (injection).
Compared with prior art, azolopyrimidines provided by the invention, its stereoisomer and its pharmaceutically Acceptable salt has better Janus kinase inhibiting activity, and it inhibits target spot to be selectively significantly better than existingization JAK2 Object is closed, and preferred compounds of the invention shows good pharmacokinetic property, has and be developed into selective JAK2 suppression The potentiality of preparation.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation Example is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common Technical staff's all other embodiment obtained without making creative work belongs to the model that the present invention protects It encloses.
Reference example 1: the synthetic route of versatile intermediates 7.
Operating procedure: step 1, the synthesis of intermediate 3.
Compound 1 (246mg, 1.0mmol), 2 (228mg, 1.0mmol) are added in DMSO (8mL), micro- under the conditions of 150 DEG C Wave reacts 2h.Reaction mixture is concentrated to dryness, and residue is purified by silica gel column chromatography (mobile phase is methylene chloride), is obtained yellow Color solid intermediate 3 (186mg, yield 42%).
1HNMR (400MHz, DMSO-d6): δ=8.97 (s, 1H), 8.17 (s, 1H), 8.08 (s, 1H), 7.87-7.81 (m, 1H), 7.54-7.46 (m, 3H), 4.10 (q, J=7.2Hz, 2H), 3.79 (s, 2H), 2.44 (s, 3H), 1.21 (t, J= 7.2Hz, 3H), 1.12 (s, 9H) .LCMS:MS Calcd.:438.6, MS Found:439.3.
Step 2, the synthesis of intermediate 4.
Into DMF (20mL) solution of compound 3 (480mg, 1.1mmol) be added potassium hydrogen persulfate (1.35g, 2.2mmol).Reaction solution is stirred overnight at room temperature, and LC-MS monitors fully reacting.Reaction mixture is concentrated to dryness, residue warp (mobile phase is methylene chloride/methanol: 30/1), obtaining yellow solid intermediate 4 (220mg, yield 43%) for silica gel column chromatography purifying. LCMS:MS Calcd.:470.6,MS Found:471.2。
Step 3, the synthesis of intermediate 5.
Into THF (60mL) solution of compound 4 (1.06g, 2.42mmol) be added sodium borohydride (184mg, 4.84mmol), reaction solution is stirred overnight at room temperature, and TLC monitors fully reacting.Reaction mixture is concentrated to dryness, residue warp (mobile phase is methylene chloride/methanol: 100/1 to 30/1), obtaining white solid intermediate 5, (622mg is received for silica gel column chromatography purifying Rate 65%).LCMS:MS Calcd.:428.5,MS Found:429.2.
Step 4, the synthesis of intermediate 6.
Into methylene chloride (20mL) solution of compound 4 (142mg, 0.33mmol) be added methane sulfonyl chloride (76mg, 0.66mmol) and 1h is stirred at room temperature in triethylamine (100mg, 0.99mmol), reaction solution, and TLC monitors fully reacting.Reaction mixture It is concentrated to dryness, residue is purified by silica gel column chromatography that (mobile phase is methylene chloride/methanol: 100/1 to 30/1), obtaining yellow Solid intermediate 6 (122mg, yield 72%).LCMS:MS Calcd.:506.6,MS Found:507.3.
Step 5, the synthesis of intermediate 7.
DBU (36mg, 0.24mmol) is added into DMF (10mL) solution of compound 5 (122mg, 0.24mmol), reaction 80 DEG C of liquid are stirred to react 1h, and LC-MS monitors fully reacting.Reaction mixture is concentrated to dryness, and residue is through prep-HPLC (NH4Ac as additive) preparative separation obtains yellow solid intermediate 7 (36.4mg, yield 39%).1HNMR(400MHz, CDCl3): δ=8.50 (s, 1H), 8.24 (s, 1H), 7.92 (dd, J=1.6Hz, 1H), 7.66 (d, J=8.0Hz, 1H), 7.55 (t, J=8.0Hz, 1H), 4.65 (m, 1H), 4.29 (s, 1H), 4.29 (t, J=8.8Hz, 2H), 3.40-3.26 (m, 5H), 1.26(s,9H)。
The synthesis of embodiment 1:I-1
Synthetic route:
Operating procedure:
Compound 7 (20mg, 0.05mmol), IA-1 (30mg, 0.15mmol) are added in trifluoroacetic acid (5mL), and system adds Heat monitors fully reactings to 100 DEG C of interior temperature reaction 12h, LC-MS.Reaction mixture is concentrated to dryness, and residue is through prep- HPLC(NH4Ac as additive) preparative separation obtains gray solid I-1 (20.2mg, yield 77%).1HNMR(400MHz, CD3OD): δ=8.34 (s, 1H), 7.97 (d, J=8.4Hz, 1H), 7.70 (s, 1H), 7.47-7.33 (m, 4H), 6.84 (d, J =8.8Hz, 2H), 4.08-3.97 (m, 4H), 2.99 (t, J=8.4Hz, 2H), 2.85 (t, J=5.2Hz, 2H), 2.67-2.56 (m, 4H), 1.80-1.68 (m, 4H), 1.08 (s, 9H) .LCMS:MS Calcd.:536.7, MS Found:537.0.
The synthesis of embodiment 2:I-2
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 70%.1HNMR (400MHz, CD3OD): δ=8.35 (s, 1H), 7.96 (d, J=8.0Hz, 1H), 7.71 (s, 1H), 7.45-7.35 (m, 4H), 6.83 (d, J=8.8Hz, 2H), 4.05- 3.97 (m, 4H), 2.97 (t, J=8.4Hz, 2H), 2.87 (t, J=5.6Hz, 2H), 2.65-2.56 (m, 4H), 2.47-2.25 (m, 4H), 1.07 (s, 9H) .LCMS:MS Calcd.:551.7, MS Found:552.3.
The synthesis of embodiment 3:I-3
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 75%.1HNMR(400MHz,CD3OD): δ=8.33 (s, 1H), 7.95 (d, J=8.0Hz, 1H), 7.73 (s, 1H), 7.44-7.35 (m, 4H), 6.82 (d, J=8.4Hz, 2H), 4.06- 3.97 (m, 4H), 3.87-3.59 (m, 4H), 2.98 (t, J=8.4Hz, 2H), 2.87 (t, J=5.6Hz, 2H), 2.45-2.28 (m,4H),1.09(s,9H).LCMS:MS Calcd.:552.7,MS Found:553.2。
The synthesis of embodiment 4:I-4
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 73%.1HNMR(400MHz,CD3OD): δ=8.37 (s, 1H), 7.97 (d, J=8.0Hz, 1H), 7.72 (s, 1H), 7.46-7.37 (m, 4H), 6.82 (d, J=8.8Hz, 2H), 4.06- 3.97 (m, 4H), 2.96 (t, J=8.0Hz, 2H), 2.88 (t, J=5.6Hz, 2H), 2.67-2.56 (m, 4H), 2.48-2.27 (m, 4H), 2.25 (s, 3H), 1.05 (s, 9H) .LCMS:MS Calcd.:565.7, MS Found:566.2.
The synthesis of embodiment 5:I-5
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 79%.1HNMR (400MHz, CD3OD): δ=8.33 (s, 1H), 7.93 (d, J=8.0Hz, 1H), 7.70 (s, 1H), 7.43-7.31 (m, 4H), 6.80 (d, J=8.0Hz, 2H), 4.07- 3.98 (m, 4H), 2.97 (t, J=8.0Hz, 2H), 2.88 (t, J=5.2Hz, 2H), 2.49-2.31 (m, 4H), 1.67-1.52 (m, 6H), 1.07 (s, 9H) .LCMS:MS Calcd.:550.7, MS Found:551.2.
The synthesis of embodiment 6:I-6
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 67%.1HNMR(400MHz,CD3OD): δ=8.34 (s, 1H), 7.95 (d, J=8.0Hz, 1H), 7.72 (s, 1H), 7.43-7.32 (m, 4H), 6.83 (d, J=8.0Hz, 2H), 4.09- 3.98 (m, 4H), 3.69-3.57 (m, 4H), 2.98 (t, J=8.0Hz, 2H), 2.93-2.89 (m, 4H), 2.85 (t, J= 5.2Hz,2H),1.07(s,9H).LCMS:MS Calcd.:600.7,MS Found:601.2。
The synthesis of embodiment 7:I-7
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 75%.1HNMR(400MHz,CD3OD): δ=8.33 (s, 1H), 7.93 (m, 1H), 7.71 (s, 1H), 7.43-7.31 (m, 4H), 6.82 (d, J=8.0Hz, 2H), 4.10 (t, J= 8.0Hz, 2H), 2.97 (t, J=8.0Hz, 2H), 2.57-2.43 (m, 6H), 2.29 (s, 3H), 1.81-1.63 (m, 5H), 1.08 (s, 9H) .LCMS:MS Calcd.:534.7, MS Found:535.3.
The synthesis of embodiment 8:I-8
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 78%.1HNMR (400MHz, CD3OD): δ=8.35 (s, 1H), 7.94 (d, J=8.0Hz, 1H), 7.72 (s, 1H), 7.45-7.31 (m, 4H), 6.86 (d, J=8.0Hz, 2H), 4.13 (t, J=8.0Hz, 2H), 3.69 (s, 2H), 2.99 (t, J=8.0Hz, 2H), 2.56-2.38 (m, 10H), 1.09 (s, 9H), 1.03(m,3H).LCMS:MS Calcd.:549.7,MS Found:550.3。
The synthesis of embodiment 9:I-9
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 71%.1HNMR(400MHz,CD3OD): δ=8.31 (s, 1H), 7.92 (d, J=8.0Hz, 1H), 7.70 (s, 1H), 7.44-7.29 (m, 4H), 6.85 (d, J=8.0Hz, 2H), 4.12 (t, J=8.0Hz, 2H), 3.69 (s, 2H), 3.51-3.37 (m, 4H), 2.97 (t, J=8.0Hz, 2H), 2.87-2.78 (m, 4H),1.09(s,9H).LCMS:MS Calcd.:570.7,MS Found:571.3。
The synthesis of embodiment 10:I-10
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 75%.1HNMR(400MHz,CD3OD): δ=8.34 (s, 1H), 7.91 (m, 1H), 7.70 (s, 1H), 7.42-7.31 (m, 4H), 6.84 (d, J=8.0Hz, 2H), 4.12 (t, J= 8.0Hz, 2H), 3.72-3.55 (m, 4H), 2.97 (t, J=8.0Hz, 2H), 2.45 (s, 2H), 1.81-1.62 (m, 5H), 1.06 (s,9H).LCMS:MS Calcd.:521.7,MS Found:522.3。
The synthesis of embodiment 11:I-11
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 71%.1HNMR(400MHz,CD3OD): δ=8.33 (s, 1H), 7.92 (d, J=8.0Hz, 1H), 7.73 (s, 1H), 7.41-7.29 (m, 4H), 6.84 (d, J=8.0Hz, 2H), 4.13 (t, J=8.0Hz, 2H), 3.69-3.65 (m, 6H), 2.97 (t, J=8.0Hz, 2H), 2.55-2.48 (m, 4H), 1.05 (s, 9H).LCMS:MS Calcd.:522.7,MS Found:523.1。
The synthesis of embodiment 12:I-12
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 68%.1HNMR(400MHz,CD3OD): δ=8.31 (s, 1H), 7.92 (d, J=8.0Hz, 1H), 7.69 (s, 1H), 7.42-7.31 (m, 4H), 6.83 (d, J=8.0Hz, 2H), 4.14 (t, J=8.0Hz, 2H), 3.67 (s, 2H), 3.03 (t, J=8.0Hz, 2H), 2.49-2.38 (m, 8H), 2.30 (s, 3H), 1.06(s,9H).LCMS:MS Calcd.:535.7,MS Found:536.3。
The synthesis of embodiment 13:I-13
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 65%.1HNMR(400MHz,CD3OD): δ=8.32 (s, 1H), 7.92 (d, J=8.0Hz, 1H), 7.70 (s, 1H), 7.40-7.29 (m, 4H), 6.83 (d, J=8.0Hz, 2H), 4.12 (t, J=8.0Hz, 2H), 3.67 (s, 2H), 2.95 (t, J=8.0Hz, 2H), 2.53-2.43 (m, 8H), 1.07 (s, 9H) .LCMS:MS Calcd.:538.7,MS Found:539.1。
The synthesis of embodiment 14:I-14
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 75%.1HNMR(400MHz,CD3OD): δ=8.32 (s, 1H), 7.90 (d, J=8.0Hz, 1H), 7.75 (s, 1H), 7.42-7.35 (m, 4H), 6.83 (d, J=8.4Hz, 2H), 4.07- 3.97 (m, 4H), 3.89-3.67 (m, 4H), 2.97 (t, J=8.4Hz, 2H), 2.82 (t, J=5.6Hz, 2H), 2.46-2.28 (m,4H),1.08(s,9H).LCMS:MS Calcd.:568.7,MS Found:569.2。
The synthesis of embodiment 15:I-15
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 73%.1HNMR(400MHz,CD3OD): δ=8.38 (s, 1H), 7.96 (d, J=8.0Hz, 1H), 7.71 (s, 1H), 7.48-7.33 (m, 4H), 6.91 (d, J=9.2Hz, 2H), 4.05 (t, J=8.4Hz, 2H), 3.12-3.15 (m, 4H), 3.01 (t, J=8.0Hz, 2H), 2.58-2.48 (m, 4H), 2.26 (s, 3H),1.07(s,9H).LCMS:MS Calcd.:521.7,MS Found:522.0。
The synthesis of embodiment 16:I-16
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 70%.1HNMR(400MHz,CD3OD): δ=8.35 (s, 1H), 7.95 (d, J=8.0Hz, 1H), 7.70 (s, 1H), 7.49-7.33 (m, 4H), 6.90 (d, J=9.2Hz, 2H), 4.09 (t, J=8.4Hz, 2H), 3.52-3.45 (m, 4H), 2.98 (t, J=8.0Hz, 2H), 2.68-2.57 (m, 4H), 1.07 (s, 9H).LCMS:MS Calcd.:507.7,MS Found:508.2。
The synthesis of embodiment 17:I-17
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 63%.1HNMR(400MHz,CD3OD): δ=8.34 (s, 1H), 7.93 (d, J=8.0Hz, 1H), 7.71 (s, 1H), 7.48-7.33 (m, 4H), 6.90 (d, J=9.2Hz, 2H), 4.13 (t, J=8.4Hz, 2H), 3.79-3.60 (m, 4H), 3.25-3.12 (m, 4H), 2.99 (t, J=8.0Hz, 2H), 1.09 (s, 9H).LCMS:MS Calcd.:508.6,MS Found:509.2。
The synthesis of embodiment 18:I-18
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 73%.1HNMR(400MHz,CD3OD): δ=8.35 (s, 1H), 7.92 (d, J=8.0Hz, 1H), 7.70 (s, 1H), 7.47-7.31 (m, 4H), 6.93 (d, J=9.2Hz, 2H), 4.12 (t, J=8.0Hz, 2H), 3.59-3.40 (m, 4H), 2.97 (t, J=8.0Hz, 2H), 1.59-1.43 (m, 5H), 1.07 (s, 9H).LCMS:MS Calcd.:506.7,MS Found:507.2。
The synthesis of embodiment 19:I-19
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 76%.1HNMR(400MHz,CD3OD): δ=8.34 (s, 1H), 7.91 (d, J=8.0Hz, 1H), 7.75 (s, 1H), 7.45-7.31 (m, 4H), 6.91 (d, J=9.2Hz, 2H), 4.13 (t, J=8.0Hz, 2H), 3.75 (m, 1H), 2.98 (t, J=8.0Hz, 2H), 2.68-2.50 (m, 4H), 2.20-2.13 (m, 4H),1.08(s,9H).LCMS:MS Calcd.:522.7,MS Found:523.2。
The synthesis of embodiment 20:I-20
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 79%.1HNMR(400MHz,CD3OD): δ=8.33 (s, 1H), 7.91 (d, J=8.0Hz, 1H), 7.70 (s, 1H), 7.49-7.33 (m, 4H), 6.93 (d, J=9.2Hz, 2H), 4.10 (t, J=8.0Hz, 2H), 2.97 (t, J=8.0Hz, 2H), 2.79-2.60 (m, 5H), 2.10-1.98 (m, 4H), 1.07 (s, 9H).LCMS:MS Calcd.:506.7,MS Found:507.2。
The synthesis of embodiment 21:I-21
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 77%.1HNMR(400MHz,CD3OD): δ=8.32 (s, 1H), 7.93 (d, J=8.0Hz, 1H), 7.71 (s, 1H), 7.47-7.32 (m, 4H), 6.91 (d, J=8.8Hz, 2H), 4.13 (t, J=8.0Hz, 2H), 3.03 (t, J=8.0Hz, 2H), 2.79 (m, 1H), 2.49-2.35 (m, 4H), 2.27 (s, 3H), 1.89-1.75(m,4H),1.05(s,9H).LCMS:MS Calcd.:520.7,MS Found:521.2。
The synthesis of embodiment 22:I-22
Synthetic route:
Operating procedure:
Operating procedure and purification process are referring to embodiment 1, yield 76%.1HNMR(400MHz,CD3OD): δ=8.33 (s, 1H), 7.91 (d, J=8.0Hz, 1H), 7.72 (s, 1H), 7.43-7.29 (m, 4H), 6.85 (d, J=8.0Hz, 2H), 4.14 (t, J=8.0Hz, 2H), 3.69 (s, 2H), 2.97 (t, J=8.0Hz, 2H), 2.51-2.43 (m, 4H), 1.67-1.52 (m, 4H),1.08(s,9H).LCMS:MS Calcd.:506.7,MS Found:507.2。
Biological test
Test case 1, JAK1, JAK2, JAK3 active testing
Compound is prepared:
Compound is dissolved in 100%DMSO, is configured to 10mM storing liquid, and -20 DEG C freeze.
Kinase reaction process:
(1) 1 × Kinase buffer is prepared.
(2) preparation of compound concentration gradient: test-compound initial concentration is 500nM, is diluted in 384source plate At the 100%DMSO solution of 100 times of final concentrations, with 3 times of diluted compounds of Precision, 12 concentration.Use dispenser Compound of the Echo 550 to purpose plate OptiPlate-384F transfer 100 times of final concentrations of 250nL.
(3) kinase solution of 2.5 times of final concentrations is prepared with 1 × Kinase buffer.
(4) add the kinase solution of 2.5 times of final concentrations of 10 μ L respectively in compound well and Positive control wells；In negative control 1 × Kinase buffer of 10 μ L of Kong Zhongjia.
(5) 1000rpm is centrifuged 30 seconds, and reaction plate oscillation is incubated at room temperature 10 minutes after mixing.
(6) mixing that the ATP and Kinase substrate of 5/3 times of final concentration is prepared with 1 × Kinase buffer is molten Liquid.
(7) ATP of 5/3 times of final concentration of 15 μ L and the mixed solution of substrate, starting reaction is added.
(8) 384 orifice plate 1000rpm are centrifuged 30 seconds, oscillation is incubated at room temperature the corresponding time after mixing.
(9) 30 μ L termination detection liquid are added and stop kinase reaction, 1000rpm is centrifuged 30 seconds, and oscillation mixes.
(10) Caliper EZ Reader reading and converting rate is used.
Data analysis:
Calculation formula:
Wherein: Conversion%_sample is the conversion ratio reading of sample；Conversion%_min: negative control Hole mean value represents the conversion ratio reading in no enzyme activity hole；Conversion%_max: Positive control wells ratio mean value, representative do not have There is compound to inhibit the conversion ratio reading in hole.
It is fitted amount effect curve:
Using the log value of concentration as X-axis, percent inhibition is Y-axis, using analysis software GraphPad Prism's 5 Log (inhibitor) vs.response-Variable slope is fitted amount effect curve, to obtain each compound to enzyme activity The IC50 value of property, calculation formula:
Y=Bottom+ (Top-Bottom)/(1+10^ ((LogIC50-X) * HillSlope))
Above-mentioned experimental result is as shown in table 2.
Table 2, compound enzyme experimental results
Note: equal product are compareed above, the compounds of this invention is same experiment condition measured value.
Conclusion: the compounds of this invention to JAK2 target spot be selectively better than positive control Baricitinib, Ruxolitinib, Fedratinib。
Test case 2, cell proliferation experiment
HEL92.1.7 cell proliferation experiment
Experimental procedure:
(1) bed board
A. cell dissociation is resuspended, is counted using automatic cell counter；
B. cell suspending liquid is diluted to required density；
C. each hole spreads 100ul cell, 37 DEG C of overnight incubations；
(2) compound is prepared
A., compound is made into 200 times of final concentration of dilute solution；
B. culture medium diluted compounds are used, 3 times of final concentration of compound is made into.Every hole adds 50ul compound, same to be added The hole of the DMSO of sample volume, which is used as, to be compareed, and 37 DEG C, 5%CO2Culture 72 hours；
(3) it detects
A. cell plates are equilibrated into room temperature；
B. every hole adds 40 μ L CellReagent shakes 2 minutes, stands 10 minutes；
C. it is detected with EnVision.
Data analysis:
(1) IC50 is calculated using GraphPad Prism 5.
(2) %Inh=(Max signal-Compound signal)/(Max signal-Min signal) x 100.
(3) Max signal is Positive control wells, the only DMSO with compound equal volume.
(4) Min signal is negative control hole, only culture medium.
TF-1 cell proliferation experiment
(1) plating cells
A. complete medium is prepared.
B. recovery cell cultivates cell.
C. cell is centrifuged, and is resuspended, and is counted, and culture plate is placed in CO by bed board2In incubator overnight.
(2) preparation and addition of compound
A., compound is configured to the stock storing liquid of 10mM with DMSO, 10mM is diluted to working concentration, gradually multiple Dilution, obtains the compound of multiple concentration gradients.
B. it is added in the tissue culture plate that is incubated overnight from pipetting 0.5ul in corresponding compound plate.
C. it is incubated for 72 hours in 37 DEG C of incubators.
(3) it detects and analyzes
A. CellTiter Glo assay detection reagent is prepared.
B. it will test reagent to be added in culture plate, mix, stand, read plate.
Inhibiting rate formula is (1- (average value of the numerical value-BLANK of the corresponding aperture)/(average value-BLANK of DMSO control Average value)) * 100%)
Curve-fitting tool (XL fit) formula is Data Analysis:(XLfit software:Fit model: Dose response one site/f (x) 205 [fit=(A+ ((B-A)/(1+ ((C/x) ^D))))])
Above-mentioned experimental result is as shown in table 3.
Table 3, cell proliferation experiment test result
Note: equal product are compareed above, the compounds of this invention is same experiment condition measured value.
Conclusion: the compounds of this invention has apparent proliferation inhibition activity to HEL92.1.7, TF-1, and inhibitory activity is better than Fedratinib、Ruxolitinib。
Test case 3, the test of the compounds of this invention pharmacokinetics
Using SD rat as animal subject, Fedratinib is given using LC/MS/MS method measurement rat oral gavage and the present invention is excellent After selecting embodiment compound, the drug concentration in its different moments blood plasma is measured, studies the compounds of this invention medicine in rat body For dynamic characteristic.
SD rat source: Shanghai Slac Experimental Animal Co., Ltd.
Administration mode: single oral gavage administration
Dosage and concentration: 25mg/kg；2mg/mL
Preparation prescription: 0.5%methylcellulose
Sample point: 5min, 15min, 30min, 1h, 2h, 4h, 8h, for 24 hours
Standard curve and Quality Control sample preparation processing: take appropriate stock solution to be diluted to 0.04 with 50% acetonitrile water, 0.10, 0.20, the standard working solution of 0.40,1.00,2.00,4.00 μ g/mL, the Quality Control working solution of 0.10,1.00,3.00 μ g/mL.Point The standard curve working solution and Quality Control working solution that 2.50 μ L are added in 47.5 μ L blank rat plasmas are not taken, are configured to containing determinand Concentration is 2.00,5.00,10.00,20.00,50.00,100.00, the mark of 200.00ng/mL is bent and concentration is 5.00,50.00 With the Quality Control sample of 150.00ng/mL, it is separately added into the acetonitrile (containing the internal standard Loratadine 5ng/mL) of 200 μ L, vortex oscillation After 3min, 15000rpm, 4 DEG C of centrifugation 15min take 100 μ L of supernatant to carry out LC-MS/MS analysis.Using8.0 Experiment with computing result.
Preferred compound pharmacokinetic parameter of the present invention is as shown in table 4.
Table 4: preferred compound pharmacokinetic parameter
Conclusion: compound of the embodiment of the present invention shows good pharmacokinetic property, compared with Fedratinib, tool There is apparent pharmacokinetic advantage.Test case 4, the compounds of this invention acute toxicity test
7 kinds of compounds (I-1, I-2, I-4, I-13, I-15, I-19 and I-20) of the present invention are chosen, and Fedratinib (positive control drug) carries out acute toxicity testing.
(1) experimental program
1., observe it and oral give what animal after the compounds such as ICR mouse Fedratinib, I-1 of the present invention occurred Toxicity symptom and death condition compare its acute toxicity.
2., solvent prepare: weigh appropriate Tween-80, with deionized water be diluted to concentration be 5% (g/v) Tween-80.
3., drug-delivery preparation: weigh required test sample respectively, with 5% Tween 80 solution be configured to concentration be 6.25, 12.50,25.00,50.00,75.00 and 100.00mg/mL (is respectively equivalent to 125,250,500,1000,1500,2000mg/ Kg) suspension.
4., administration route: the administration route of test sample and vehicle control group (0.5% Tween-80) is to be administered orally.
5., administration frequency: single-dose, be administered before fasting overnight.
6., administration capacity: 20mL/kg.
General symptom observation: it observed 1 time respectively within the administration same day about 0.5,1,2,4,6 hour after being administered for the first time；Observation Phase the 2nd~6 day, observation 2 times daily, each 1 time of upper and lower noon.
Observed content includes but is not limited to: general status, behavioral activity, gait posture, eye, mouth, nose, gastrointestinal tract, skin Skin coat, urogenital tract.
(2) it statisticallys analyze
Weight data is indicated with mean ± standard deviation, and is examined and single factor test variance using comparison among groups using Levene`s Analysis if display is variant, then is examined using Dunnet t.
(3) experimental result
Choose 7 kinds of compounds and Fedratinib (positive control drug) of the present invention such as the above-mentioned acute poison of carry out Property experiment.Experimental result is shown in Table 5.
In MTD test, animal is investigated to the tolerance situation of drug, and it is most that dosage, which reaches animal frequency, when dying on one's deathbed Big dosis tolerata.
Acute toxicity testing result is administered in the compounds such as table 5:I-1 and Fedratinib single oral
Note: MTD: maximal tolerance dose.
The result shows that: the MTD (maximal tolerance dose) of the compounds of this invention I-1, I-15, I-20 are all larger than in above-mentioned tested material 2000mg/kg, acute toxicity is well below Fedratinib；The MTD value of compound I-2, I-4, I-13, I-19 are all larger than or wait In 1000mg/kg, safety is better than Fedratinib.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Claims (8)

1. a kind of pyrrolin miazines selectivity JAK2 inhibitor or its pharmaceutically acceptable salt shown in formula I,
Wherein:
X is O or is not present；
Y is O, S, SO2Or NR；
W is N or CH；
R is hydrogen, C1~6Alkyl, C2~6Alkenyl, C2~6Alkynyl, C1~6Alkoxy or C1~6Carbonyl；
M is 0,1,2,3,4,5 or 6；
N is 0,1 or 2.
2. pyrrolin miazines selectivity JAK2 inhibitor as described in claim 1 or its pharmaceutically acceptable salt, It is characterized in that, wherein:
X is O or is not present；
Y is O, S, SO2Or NR；
W is N or CH；
R is hydrogen or C1~4Alkyl；
M is 0,1,2 or 3；
N is 0 or 1.
3. pyrrolin miazines selectivity JAK2 inhibitor as described in claim 1 or its pharmaceutically acceptable salt, It is characterized in that, wherein:
X is O or is not present；
Y is O, S, SO2Or NR；
W is N or CH；
R is hydrogen or methyl；
M is 0,1 or 2；
N is 0 or 1.
4. pyrrolin miazines selectivity JAK2 inhibitor as described in claim 1 or its pharmaceutically acceptable salt, It is characterized in that, being selected from following compound:
5. it is a kind of prepare as the described in any item pyrrolin miazines selectivity JAK2 inhibitor of Claims 1 to 4 or its The method of pharmaceutically acceptable salt, it is characterised in that include the following steps:
(1) compound 1 and 2 is made 3 through condensation reaction；
(2) compound 3 aoxidizes to obtain compound 4 using potassium acid sulfate；
(3) compound 4 obtains compound 5 through sodium borohydride reduction；
(4) the obtained versatile intermediates 7 of cyclization reaction occur after sulfonyl methane chlorine activation for the hydroxyl of compound 5；
(5) compound 7 is reacted with general formula IA compound condensation is made final product I.
6. as the described in any item pyrrolin miazines selectivity JAK2 inhibitor of Claims 1 to 4 or its pharmaceutically may be used Purposes of the salt of receiving in the drug of preparation prevention or treatment and JAK related disease.
7. purposes as claimed in claim 6, it is characterised in that it is described include with JAK related disease organ-graft refection, lupus, Multiple sclerosis, adolescent arthritis, psoriasis, ulcer disease colitis, Crohn's disease, is exempted from rheumatoid arthritis self Epidemic disease thyroid disease, ox-hide moss, skin itch, atopic dermatitis, asthma, rhinitis, hepatitis B, hepatitis C, varicella blister It is exanthema virus, type-1 diabetes mellitus and diabetic complication, Alzheimer disease, scheroma, myelofibrosis, piastrenemia, red Cytosis, leukaemia, Huppert's disease, prostate cancer, kidney, liver cancer, film gland cancer, gastric cancer, breast cancer, lung cancer, head Neck cancer, thyroid cancer, glioblastoma, melanoma, lymthoma, leukaemia, cutaneous T-cell lymphomas or cutaneous T-cell Lymthoma.
8. a kind of composition, the composition includes therapeutically effective amount such as the described in any item pyrrolin of Claims 1 to 4 Miazines selectivity JAK2 inhibitor or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier.
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